Avian herpesvirus-A antigen precursor gene

ABSTRACT

A gene encoding for A antigen precursor, derived from a Avian herpesvirus, is described. The gene is preferably isolated from a Marek&#39;s disease herpesvirus virus (MDHV) and is approximately 1.8-1.9 kbp, found on a DNA fragment of about 2.35 kbp in size. The gene can be used as a building block for vaccines, directly as a diagnostic hybridization probe, to facilitate preparation of diagnostic test kits for MDHV by antigen production and subsequent antibody production, and as a potential source of regulatory elements such as signal peptides or promoters for use with other proteins.

This is a continuation of application Ser. No. 07/526,790 filed on May17, 1990, now abandoned, which is a continuation of application Ser. No.07/041,974, filed Apr. 24, 1987, now abandoned.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a substantially pure fragment of anAvian herpesvirus genome containing the gene for A antigen precursorpolypeptide pr47 or a subfragment thereof which is the unglycosylatedprecursor to A antigen (gp57-65). In particular the present inventionrelates to an about 2.35 kbp fragment or subfragment of DNA from aMarek's disease herpesvirus which contains the gene that encodes for Aantigen precursor pr47.

(2) Prior Art

Marek's disease (MD) is a lymphoproliferative disease of chickens causedby Marek's disease herpesvirus (MDHV), which results in T-cell lymphomasand peripheral nerve demylination (Marek, J., Multiple Nervenetzuendung(Polyneuritis) bei Huchnern. Dtsch. Tieraerztl. Wochenschr. 15:417-421(1907); Pappenheimer, A. M., L. C. Dunn, and V. Cane, J. Exp. Med.49:63-86 (1929); and Pappenheimer, A. M., L. C. Dunn, and S. M. Seidlin,J. Exp. Med. 49:87-102 (1929)). The disease was a major cause ofeconomic loss ($200 million/year) to the poultry industry until theearly 1970's (Purchase, H. G., Beltsville Symp. Agri. Res. 1:63-81(1977)), when a live vaccine was developed from the antigenicallyrelated apathogenic herpesvirus of turkey (HVT) (Okazaki, W., H. G.Purchase, and B. R. Burmester, Avian Dis. 14:413-429 (1970)).

The ability of HVT to protect chickens against infection by MDHV may bedue to an antigenic relationship between HVT and MDHV. At least sixantigenically active viral proteins are common between MDHV and HVT(Ikuta, K. S., S. Ueda, S. Kato, and K. Hirai, J. Gen Virol. 64:961-965(1983); and Silva, R. F., and L. F. Lee, Virology 136-307-320 (1984)).One of these antigenic proteins, the prominent MDHV A antigen (MDHV-A),was the first MDHV-HVT common antigen to be characterized on a physical,chemical and molecular basis (Glaubiger, C., K. Nazerian, and L. F.Velicer., J. Virol. 45:1228-1234 (1983); Long, P. A., K. Y. Parveneh,and L. F. Velicer, J. Virol. 15:1182-1192 (1975); and Long, P. A., J. L.Clark, and L. F. Velicer, J. Virol. 15:1192-1201 (1975)). MDHV-A is aglycoprotein with an apparent molecular weight of 57-65,000 daltonsreferred to as (gp57-65) in its fully glycosylated form (Glaubiger, C.,K. Nazerian, and L. F. Velicer, J. Virol. 45:1228-1234 (1983); andIsfort, R. J., R. A. Stringer, H. J. Kung, and L. F. Velicer, J. Virol.57:464-474 (1986)). A combination of cell-free translation, pulse-chase,and tunicamycin inhibitor studies have shown that MDHV-A is synthesizedas a 47,000 dalton precursor polypeptide that apparently undergoescleavage to remove a small signal peptide (Isfort, R. J., R. A.Stringer, H. J. Kung, and L. F. Velicer, J. Virol. 57:464-474 (1986)).The resulting 44,000 dalton polypeptide undergoes glycosylation andsecretion from the cell in a precisely programmed manner (Isfort, R. J.,R. A. Stringer, H. J. Kung, and L. F. Velicer, J. Virol. 57:464-474(1986)). While MDHV-A is primarily secreted from infected cells (Isfort,R. J., R. A. Stringer, H. J. Kung, and L. F. Velicer, J. Virol.57:464-474 (1986)), there is mounting evidence that a small amount isalso associated with the plasma membrane in a specific manner (Ikuta, K.S., S. Ueda, S. Kato, and K. Hirai, J. Gen. Virol. 64:2597-2610 (1983);Nazerian, K., J. Gen. Virol. 21:193-195 (1973)). Not only is its role inthe immunoprevention by HVT still unclear, it could have a role in thepathogenesis of MD since it has been recently postulated to play animmunoevasive role in protecting virus-producing cells of the featherfollicle epithelium (Isfort, R. J., R. A. Stringer, H. J. Kung, and L.F. Velicer, J. Virol. 57:464-474 (1986)). Furthermore any virus-encodedglycoprotein should be a candidate for causing the early stageimmunosuppression that is reported to occur after MDHV infection and maybe one of the key events that lead to neoplasia (Payne, L. W., Biologyof Marek's disease virus and the herpesvirus of turkeys, pp. 347-431. InB. Roizman (ed.), The herpesviruses, vol. 1. Plenum Press (1982));especially in view of the observation that various virus particles,either infectious or non-infectious, inhibit mitogenic responses(Wainberg, M. A., B. Beiss, and E. Israel, Avian Dis. 24:580-590(1980)).

OBJECTS

It is an object of the present invention to provide the gene thatencodes for the precursor protein of antigen MDHV-A. It is further anobject of the present invention to provide the gene for furthermolecular characterization of the protein, and for producing expressionproducts of the MDHV-A gene in order to better assess its role(s) in theimmunosuppression, immunoevasion and/or immunoprotection processes.These and other objects will become increasingly apparent by referenceto the following description and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show a SDS-PAGE analysis of cell-free translationproducts of mRNA hybrid-selected using 14 individual pBR328 MDHV DNAclones immobilized on nitrocellulose. FIG. 1A shows a direct analysis ofthe products of MDHV-specific mRNAs that were hybrid-selected andcell-free translated. Lanes 1-15 show translation of cytoplasmic RNAisolated 72 hours post infection (PI) from MDHV-infected cells andselected using pBR328 clones 1-15; lane 16 (INF) shows translation ofunselected cytoplasmic RNA isolated 72 hours PI from MDHV-infected DEF;lane 17 (CON) shows translation of unselected cytoplasmic RNA isolatedfrom uninfected DEF; lane 18 (RNA) shows translation with no added RNA.Ten microliters of each translation mixture was analyzed per lane todisplay all the polypepetides produced. Samples were analyzed on 10%SDS-PAGE and the autoradiogram of the dried gel was prepared byfluorographic exposure for 2 days. FIG. 1B shows immunoprecipitation andSDS-PAGE analysis of the products of hybrid-selected and cell-freetranslated MDHV specific mRNAs. Lanes 1-17 of FIG. 1B are the same as inA, but the translation products were immunoprecipitated with RαA sera.Forty microliters of each translation reaction mixture was subjected toimmunoprecipitation with 2 microliters of RαA. Samples were analyzed on10% SDS-PAGE, and the autoradiogram of the dried gel was prepared byfluorographic exposure for 5 days. Clone 15 is the only one tohybrid-select.

FIGS. 2A and 2B show a further analysis of clone pBR328-15 that selectsMDHV-A mRNA. FIG. 2A shows a restriction map of pBR328-15. FIG. 2B showscell-free translation using subfragments of pBR328-15 followed byimmunoprecipitation with RαA sera. Hybrid-selections, cell-freetranslation, immunoprecipitation, SDS-PAGE and fluorography wereperformed as in FIG. 1, with fluorographic exposures for 7 days. Onlythe 2.35 kbp fragment hybrid-selects the messenger RNA encoding for Aantigen.

FIG. 3 shows a southern blot analysis of Bam Hl-digested MDHV DNA probedwith either (A) total MDHV DNA or (B) the 2.35 Eco Rl-Pvu II MDHV DNArestriction fragment containing the MDHV-A coding region. DNA isolation,restriction enzyme digestion, blotting, hybridization, andnick-translations were performed as described in the specification. Thehybridization was specific for Bam Hl B fragment (18.3 kbp).

FIG. 4A is a map of Bam Hl B fragment showing orientation andapproximate position of the MDHV-A antigen coding region as determinedby both hybrid-selection and northern blot analysis. Bothhybrid-selection and Northern and Southern blot analysis was performedas described in the specification. The 2.35 kbp fragment containing theMDHV-A coding region is in the middle of the fragment.

FIG. 4B is a fine structure mapping of the MDHV-A gene. The entireMDHV-A coding sequence of approximately 1.8-1.9 kbp in size (between thesolid arrows and broken arrow) has been localized within the 2.35 kbpPvu II-EcoRI segment of the MDHV Bam HI B fragment. Orientation andlimits were determined by a combination of Sl nuclease, Northernblotting, and RNAse A/T₁ protection experiments using probes I-III. Slnuclease assays (using probe I) indicate two major transcriptioninitiation sites (solid arrows). RNase A/T₁ protection studies using SP6generated RNA probes II and III were used in combination with Slnuclease analysis to map the 3' end of the MDHV-A coding sequence(dashed arrow). Restriction enzymes used in subcloning and generation ofprobes were: BamHI (BI), Bgl II (BII), Kpn I (K), Ssp I (S), Nco I (N),EcoRV (V), EcoRI (E), Pvu II (P), and Sma I (SI).

FIG. 5 shows a northern blot analysis of control and infected totalcellular RNA using the 2.35 Eco Rl-Pvu II fragment as a nick-translatedprobe for hybridization. A total of 10 micrograms of RNA was loaded perlane and electrophoresed, blotted and probed as described in thespecification. The exposure was for 1 hour. The messenger RNA of Aantigen is about 1.8-1.9 kbp in size.

GENERAL DESCRIPTION

The present invention relates to a substantially pure EcoR₁ -PvuIIendonuclease digestion gene-containing fragment of an Avian herpesvirusgenome containing the gene for A antigen precursor polypeptide and asubfragment of the fragment.

The present invention also relates to a method for removing the genecoding for A antigen from DNA of an Avian herpesvirus which comprises:digesting the herpesvirus DNA or a fraction thereof with EcoR₁ -PuvIIendonucleases to provide a segment which contains the gene for Aantigen.

The gene encoding the glycoprotein MDHV-A antigen's unglycosylatedprecursor polypeptide pr47 was delineated using Northern blot analysisand hybrid-selection of its mRNA with cloned MDHV DNA, cell freetranslation of the mRNA and immunoprecipitation of the polypeptide. Theresulting piece of DNA with strongly positive hybrid-selection resultswas the 2.35 kbp Pvu II-EcoRI restriction fragment localized to thecenter of the 18.3 kbp MDHV Bam HI B fragment of the total virus genome.The much lower level of hybrid-selection and detection of mRNA inNorthern blot analysis by the adjacent 2.4 kbp ECO RI-Sma I fragmentinitially suggested it may contain a very small part of the MDHV-A gene,if any at all. It is now clear that the 3' end of the gene for MDHV-A iswithin the 2.35 kbp fragment, suggesting these borderline results aredue to read-through transcription to yield a small amount of large mRNAspanning the junction between the two fragments. The localization wasspecific since no other small restriction subfragment of the larger BAMHI B fragment was able to hybrid-select MDHV-A mRNA and the gene mappedonly in the Bam HI B fragment of the total virus genome. Northern blotanalysis confirmed the localization of the MDHV-A gene primarily on the2.35 kbp fragment and detected its mRNA as a 1.8 k daltons species, asize consistent with encoding a 47 kd polypeptide. This then is thefirst disclosure of a MDHV gene being mapped to the MDHV viral genome.This development opens the way for the use of recombinant DNA technologyto study the nature of the gene encoding a secreted virus-specificglycoprotein that could possibly be involved in immunoprevention,immunosuppression and/or immunoevasion, immune phenomena known orspeculated to be involved in this oncogenic herpesvirus system.

SPECIFIC DESCRIPTION MATERIALS AND METHODS

Cells and viruses. The preparation, propagation and infection of smallscale duck embryo fibroblast (DEF) cell culture with MDHV was generallyas first reported by Glaubiger et al. (Glaubiger, C., K. Nazerian, andL. F. Velicer, J. Virol. 45: 1228-1234 (1983) and more recently byIsfort et al (Isfort, R. J., R. A. Stringer, H. J. Kung and L. F.Velicer, J. Virol. 57:464-474 (1986). One exception was that 0.2% calfserum was present in the maintenance media, as previously reported(Isfort, R. J., R. A. Vrable, K. Nazerian, J. J. Kung and L. F. Velicer,Gene identification and molecular characterization of the Marek'sdisease virus A antigen, p. 130-147. In B. W. Calnek and J. L. Spencer(eds.), Proc. Int. Symp. Marek's Dis. The American Association of AvianPathologists, Inc., Kennett Square, Pa. (1985)). MDHV strain GA (Long,P. A., J. L. Clark, and L. F. Velicer, J. Virol. 15:1192-1201 (1975))was used at the 26th passage level.

DNA preparation, gel electrophoresis and Southern blotting. MDHV DNA inthe form of 14 pBR328 and 14 lambda clones was a gift from Dr. C. Gibbs(Gibbs, C., K. Nazerian, L. F. Velicer, and H. J. Kung, Proc. Natl.Acad. Sci. USA 81:3365-3369 (1984), currently at Rockefeller University.The MDHV Bam Hl library was a gift from Dr. M. Nonoyama, ShowaUniversity Research Institute (Fukuchi, K., M. Sudo, Y.-S. Lee, A.Tanaka, and M. Nonoyama., J. Virol. 51:102-1009 (1984)). The MDHV DNA ineach clone was propagated in bacteria and then extracted by standardmethods (Maniatis, T., E. Fritsch, and J. Sambrook., Molecular cloning.Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982)). The DNA(1 microgram from each clone) was bound to nitrocellulose forhybrid-selection using the method of Parnes et al (Parnes, J. R., B.Velan, A. Felsenfeld, L. Ramanathan, V. Ferrini, E. Apellka, and J. G.Seidman, Proc. Natl. Acad. Sci. USA 78:2253-2257 (1981)).

For further analysis, the entire MDHV genome, clone pBR328-15 and theMDHV Bam Hl clone B were digested with the appropriate restrictionenzyme(s) using the conditions recommended by the manufacturer (BethesdaResearch Laboratories located in Gaithersburg, Md.). The digests werethen analyzed by gel electrophoresis at 4 volts/cm until the leading dyefront was 3/4 of the way down the gel. The gel was then stained in 0.5ug/ml of ethidium bromide solution for 1 hour and photographed underultraviolet light. Southern transfer of the electrophoresed DNA wasperformed according to the methods of Southern (Southern, E. M., J. Mol.Biol. 98:503-517 (1975)). Nick-translations of DNA fragments to be usedas probes were performed according to the methods of Rigby et al (Rigby,P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. J. Mol. Biol.113-237-251 (1977)). Hybridization of the ³² P-labeled probes to thenitrocellulose bound restriction fragments resulting from Southernblotting was performed according to the method of Maniatis et al.(Maniatis, T., E. Fritsch, and J. Sambrook, Molecular Cloning. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982)).

Isolation of cellular RNA. The method of RNA isolation was that ofFavaloro et al (Favaloro, J., R. Treisman, and K. Kamen., Meth. Enzymol.65:718-749 (1980)). This method was modified so that the isolated RNAwas not treated with DNase prior to the final ethanol precipitation stepand the RNA was extracted five times with phenol/chloroform to removeall traces of vanadyl ribonucleotides (New England Nuclear located atBoston, Mass.).

Hybrid selection of MDHV specific mRNA. The method for hybrid-selectionwas that of Paterson et al (Paterson, B. M., B. E. Roberts, and E. L.Kuff., Proc. Natl. Acad. Sci. USA 74:4370-4374 (1977)), with the time ofhybridization and the concentration of RNA modified to achieve maximalselection. Briefly, hybridization was carried out for 3 hours using 50micrograms of total cellular RNA per filter in a 100 microliter volume.

Cell-free translation. Cell-free translation in rabbit reticulocytelysates was performed using the methods of Jackson and Hunt (Jackson, R.J., and T. Hunt., Meth. Enzymol. 96:50-74 (1983)), as already reportedin preliminary studies in the MDHV system (Isfort, R. J., R. A. Vrable,K. Nazerian, H. J. Kung, and L. F. Velicer., Gene identification andmolecular characterization of the Marek's disease virus A antigen, p.130-147. In B. W. Calnek and J. L. Spencer (eds.), Proc. Int. Symp.Marek's Dis. The American Association of Avian Pathologists, Inc.,Kennett Square, Pa. (1985); and Isfort, R. J., R. A. Stringer, H. J.Kung, and L. F. Velicer, J. Virol. 57:464-474 (1986)). In the presentinvention translation after hybrid-selection was performed using theselected RNA with 10 micrograms of calf liver RNA added as carrier.

RNA gel electrophoresis and Northern blotting. Electrophoresis of RNAwas performed according to the methods of Lehrach et al (Lehrach, E., D.Diamond, J. M. Wozney, and H. Boedtker., Biochemistry 16:4743-4751(1977), as outlined by Maniatis et al (Maniatis, T., E. Fritsch, and J.Sambrook., Molecular cloning. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1982)). Northern blotting after electrophoresis wasperformed by transfer of formaldehyde-denatured RNA to nitrocelluloseaccording to the method outlined by Maniatis et al (Maniatis, T., E.Fritsch, and J. Sambrook., Molecular Cloning. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1982)). Nick translation andhybridization with ³² P labeled probe was as described above forSouthern blot analysis.

Immunoprecipitation with RαA. Immunoprecipitations were performedaccording to the methods of Witte and Wirth (Witte, O. N., and D. F.Wirth., J. Virol. 29:735-743 (1979)), as adopted to the MDHV system byGlaubiger et al (Glaubiger, C., K. Nazerian, and L. F. Velicer, J.Virol. 45:1228-1234 (1983) and used by Isfort et al (Isfort, R. J., R.A. Stringer, H. J. Kung, and L. F. Velicer., J. Virol. 57:464-474(1986)). The RαA sera used to precipitate MDHV-A was prepared aspreviously described (Long, P. A., J. L. Clark, and L. F. Velicer, J.Virol. 15:1192-1201 (1975)) and has been used extensively for thatpurpose before (Glaubiger, C., K. Nazerian, and L. F. Velicer, J. Virol.45:1228-1234 (1983); and Isfort, R. J., R. A. Stringer, H. J. Kung, andL. F. Velicer., J. Virol. 57:464-474 (1986)).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Discontinuous stack SDS-PAGE was performed according to the method ofLaemmli (Laemmli, U.K., Nature 227:680-685 (1970)), as adapted to theMDHV system by Glaubiger et al (Glaubiger, C., K. Nazerian, and L. F.Velicer, J. Virol. 45:1228-1234 (1983)), with the previously reportedmodification (Isfort, R. J., R. A. Stringer, H. J. Kung, and L. F.Velicer, J. Virol. 57:464-474 (1986)): (1) all samples were boiled insample buffer containing 2% SDS and 5% 2-mercaptoethanol prior to gelloading, (2) electrophoresis was carried out at a constant current of 30mA until the marker dye ran off the gel. Standard molecular weightmarkers included ¹⁴ C-labeled phosphorylase B (92.5 kd), bovine serumalbumin (69 kd), ovalbumin (46 kd), and carbonic anhydrase (30 kd)(NEN). Molecular weights were calculated by interpolation betweenstandard proteins by the method of Weber and Osborn (Weber, K., and M.Osborn, J. Biol. Chem. 244:4405-4412 (1969)). Fluorography was performedaccording to the methods of Bonner and Laskey (Bonner, W. M., and R. A.Laskey, Eur. J. Biochem. 46:83-88 (1974)). Autoradiography of allsamples was carried out at -70° C.

RESULTS

In previous cell-free translation studies, it was established that theMDHV-A is synthesized as a 47,000 precursor polypeptide (pr47)immunoprecipitatable with RαA (Isfort, R. J., R. A. Stringer, H. J.Kung, and L. F. Velicer (J. Virol. 57:464-474 (1986)). Therefore, it waspossible to combine hybrid-selection with these methods to identify thegene encoding MDHV-A from a partial genomic library of MDHV DNA (Gibbs,C., K. Nazerian, L. F. Velicer, and H. J. Kung, Proc. Natl. Acad. Sci.USA 81:3365-3369 (1984)).

Hybrid-selection was performed using RNA preparations isolated at thetime of maximal A antigen synthesis (Isfort, R. J., R. A. Stringer, H.J. Kung, and L. F. Velicer (J. Virol. 57:464-474 (1986)). For theinitial hybrid-selection, both the plasmid pBR328 and the lambda MDHVlibraries were used (Gibbs, C., K. Nazerian, L. F. Velicer, and H. J.Kung, Proc. Natl. Acad. Sci. USA 81:3365-3369 (1984)). These librariescontain approximately 30% and 50% of the MDHV genome respectively(Gibbs, C., K. Nazerian, L. F. Velicer, and H. J. Kung, Proc. Natl.Acad. Sci. USA 81:3365-3369 (1984); C. Gibbs, unpublished data). UsingDNA pools of either 14 MDHV pBR328 clones or 14 lambda MDHV clones,hybrid-selection was performed under stringent conditions of 65%formamide and 50° C. Washes to remove all non-hybridized RNA wereperformed at 65° C. and 0.15M sodium chloride. After direct SDS-PAGEanalysis of the cell-free translation reaction mixture, approximately 18to 20 proteins and 10 to 12 proteins, respectively, were produced bytranslation of mRNA selected by both the lambda and pBR328 cloned MDHVDNA pools (Isfort, R. J., R. A. Vrable, K. Nazerian, H. J. Kung and L.F. Velicer., Gene identification and Molecular characterization of theMarek's disease virus A antigen, p. 130-147. In B. W. Calnek and J. L.Spencer (eds.), Proc. Int. Symp. Marek's Dis. The American Associationof Avian Pathologists, Inc., Kennett Square, Pa. (1985)). The backgroundproteins made from uninfected DEF mRNA non-specifically binding to MDHVDNA was zero for the pBR328 clone pool. However, the lambda cloned DNApool apparently selected mRNA for three DEF proteins (R. J. Isfort,unpublished data). This selection may result from the way the phagerecombinant library was made. The pBR328 clones were constructed bycloning purified MDHV Eco Rl fragments (Gibbs, C., K. Nazerian, L. F.Velicer, and H. J. Kung., Proc. Natl. Acad. Sci. USA 81:3365-3369(1984)), which results in no insertion of duck embryo fibroblast DNAsequences. However, the lambda clones were prepared by Mbo 1 partialdigest of the MSB-1 chicken lymphoid tumor cell line (Hughes, S. H., E.Stubblefield, K. Nazerian, and H. E. Varmus, Virology 105:234-240(1980)) DNA followed by insertion into the lambda cloning vector. Clonescontaining MDHV genome were selected using purified MDHV DNA as thehybridization probe (Gibbs, C., K. Nazerian, L. F. Velicer, and H. J.Kung., Proc. Natl. Acad. Sci. USA 81:3365-3369 (1984)). Since thelibrary was constructed in this manner, any chicken cellular DNA next tointegrated MDHV DNA (Hughes, S. H., E. Stubblefield, K. Nazerian, and H.E. Varmus, Virology 105:234-240 (1980)) may be carried into the lambdaclone. This could result in hybrid-selection of DEF mRNA (homologous tothe inserted chicken DNA) and subsequent translation of any proteinencoded on the mRNA. Alternatively, the lambda clones may contain MDHVsequences sharing homology with the selected DEF mRNA. The absence ofthese background proteins, using mRNA selected by the pBR328 clones, maybe due to either the incompleteness of the pBR328 MDHV library relativeto the lambda library, or the lack of CEF sequences homologous to theDEF mRNA.

When these same cell-free translation reaction mixtures were analyzed byimmunoprecipitation, MDHV-A's pr47 (Isfort, R. J., R. A. Stringer, H. J.Kung, and L. F. Velicer, J. Virol. 57:464-474 (1986)) wasimmunoprecipitated with the RαA sera from the two mixtures receivinginfected cell RNA (Isfort, R. J., R. A. Vrable, K. Nazerian, J. J. Kungand L. F. Velicer, Gene identification and molecular characterization ofthe Marek's disease virus A antigen, p. 130-147. In B. W. Calnek and J.L. Spencer (eds.), Proc. Intl. Symp. Marek's Dis. The AmericanAssociation of Avian Pathologists, Inc., Kennett Square, Pa. (1985)),indicating that the pBR328 and lambda cloned MDHV DNA pools both selecta message encoding the MDHV-A polypeptide. The inability to visualizepr47 directly without immunoprecipitation analysis of the cell-freetranslation products is due to it comigrating with, and being obscuredby, a background protein which migrates at the 40-50 kd region.

The above preliminary results indicate that both the pBR328 and lambdaclones contain DNA sequences encoding for MDHV-A pr47. For further geneidentification the 14 available MDHV pBR328 clones were usedindividually to hybrid-select the MDHV-A mRNA. The DNA of one clone,numbered 13 for identification in this study, was lost, explaining thegap in numbering of the clones. The direct SDS-PAGE analysis of thecell-free translated proteins whose mRNAs were hybrid-selected by DNAsequences contained in each clone is shown in FIG. 1A. At least 10-12different proteins result from translation of mRNA selected by the 14clones. One of the more interesting pBR328 clones is clone number 11(pBR328-11), which selects mRNA encoding for at least nine differentproteins. It should be noted that apparently many of the mRNAs selectedfor by pBR328-11 appear to be selected by other pBR328 MDHV clones (FIG.1A), since similar proteins are seen in other lanes as well.

Immunoprecipitation analysis of these cell-free translation productswith RαA showed that MDHV pBR328 clone number 15 (pBR328-15) contains aDNA sequence which selected for the mRNA of MDHV-A pr47 (FIG. 1B).Another polypeptide immunoprecipitable with RαA and migrating at about30-36 kd in the same lane on the gel (FIG. 1B) may be either a productof premature translation termination, or a protein encoded by atranscript colinear with a portion of the MDHV-A mRNA and containing anantigenic domain recognizable by RαA. As already reported (Isfort, R.J., R. A. Vrable, K. Nazerian, H. J. Kung and L. F. Velicer. Geneidentification and molecular characterization of the Marek's diseasevirus A antigen, p. 130-147. In B. W. Calnek and J. L. Spencer (eds.)Proc. Int. Symp. Marek's Dis. The American Association of AvianPathologists, Inc., Kennett Square, Pa. (1985)), and will be seen insubsequent figures, this molecule is also made every time pr47 issynthesized by cell-free translation of MDHV-specific mRNA.

Clone pBR328-15 contains two MDHV Eco Rl fragments, a 6.8 kbp fragmentand a 1.5 kbp fragment (FIG. 2A). When each of the two cloned MDHV DNAinserts and the 4.9 kbp vector DNA were tested for their ability toselect MDHV-A mRNA, it was discovered that the 6.8 kbp MDHV Eco Rlfragment contained the genomic coding sequence for MDHV-A (FIG. 2B, leftside). This is based on the presence of the dark band of pr47 just underthe more gray background smear occurring in these gels. Once again the30-36 k molecule is detected in the same lane. While these two bands arenot as apparent on the left side of FIG. 2B compared to previous andsubsequent data, apparently due to less than optimal efficiency ofselection and/or translation this one time, the correctness of thesedata is confirmed by their repetition and extension in the nextexperiment and subsequent experiments.

Upon further digestion with the restriction enzyme Pvu II, the 6.8 kbpfragment could be divided into three subfragments of 0.2, 2.35, and 4.4kbp as mapped and illustrated in FIG. 2A. When these three fragmentswere isolated and used for hybrid-selection, the results in the rightside of FIG. 2B were obtained. It is clear that the fragment whichcontains the coding region for MDHV-A is the terminal Eco Rl-Pvu II 2.35kbp fragment (FIG. 2B). Northern blot analysis with the same threesubfragments confirmed this localization of the MDHV-A gene, only the2.35 kbp subfragment detected the MDHV-A mRNA, which is described below.Note that the mRNA for the 30-36 kd protein was also hybrid-selected bythis small fragment.

Since the 2.35 kbp subfragment that selects for the A antigen mRNA liesat the end of the pBR328 6.8 kbp Eco Rl fragment, it is possible thatthe entire gene for DHV-A is not encoded on this fragment. Since noother pBR328 clone was able to select message for the A antigen (FIG.1B), it was necessary to obtain a more complete library of the MDHVgenome for further gene identification studies. Fortunately, during thecourse of this study a nearly complete library was created (Fukuchi, K.,M. Sudo, Y. S. Lee, A. Tanaka, and M. Nonoyama. J. Virol. 51:102-109(1984)) and the Bam Hl DNA fragments were made available to us. In orderto identify the Bam Hl fragment which corresponds to the MDHV pBR328clone that we have identified as encoding MDHV-A, a Bam Hl digest ofpurified MDHV DNA was separated by gel electrophoresis, Southernblotted, and used to hybridize with a probe of the labeled 2.35 kbp EcoRl-Pvu II subfragment of pBR328-15. As can be seen in FIG. 3, only theBam Hl fragment B contained the sequence corresponding to the 2.35 kbpEco Rl-Pvu II subfragment.

In order to determine where in the large (18.3 kbp) Bam Hl fragment Bthe genetic information of the 2.35 kbp Eco Rl-Pvu II sub fragment lies,fragment B was first digested with several restriction endonucleases andphysically mapped (FIG. 4). The restriction digests were gelelectrophoresed, Southern blotted and hybridized to a radioactive probeof the Eco Rl-Pvu II 2.35 kbp subfragment of pBR328-15 (FIG. 2A).Following this, the blot was then hybridized to a radioactive probe ofcomplete 6.8 kbp Eco Rl fragment. The combined information served todetermine the orientation and directionality of the 6.8 kbp fragment andits 2.35 kbp subfragment in Bam HI B. As is seen in the resultant map(FIG. 4), the 2.35 kbp Eco RI-Pvu II subfragment lies in the center ofBam Hl B fragment. Thus the adjacent sequences are available todetermine if any of the MDHV-A gene lies adjacent to the 2.35 kbp EcoRl-Pvu II subfragment known to contain at least part of the gene.

In order to delineate the extent of the A antigen gene in the Bam Hl Bfragment, the other restriction fragments of Bam Hl B shown in FIG. 4Awere isolated and used to hybrid-select the MDHV-A mRNA. From thesestudies it appears that a 2.4 kbp Eco Rl-Sma l subfragment adjacent tothe 2.35 kbp Eco Rl-Pvu II subfragment was also able to hybrid-selectthe MDHV-A mRNA (FIG. 4A), although this selection was not as stronglypositive as with the 2.35 kbp Pvu II-Eco Rl subfragment. However, otherthan these two subfragments, no other DNA sequences in the entire Bam HlB fragment were able to hybrid-select MDHV-A mRNA, thereby delineatingthe approximate limits of the A antigen coding region.

To complete this initial analyses of the MDHV-A gene Northern blotanalysis was performed using the same RNA preparations used above forhybrid selection. In this case the 2.35 kbp Pvu II-Eco Rl fragment, theone most strongly positive in hybrid-selection was nick-translated andused as a probe. It detected an mRNA band migrating just below theposition of 18S RNA marker (FIG. 5), a size of approximately 1.8 kbp.When all the other subfragments of the Bam Hl B fragment were used asprobes for Northern blot analysis none of them detected the same 1.8 kbpmRNA on short exposure of the gel to X-ray film. The 2.4 kbpEcoRl-Smalfragment was very weakly positive in detecting the same mRNA, and thenonly upon long exposure (FIG. 4).

DISCUSSION

The purpose of this invention was to identify the location of the codingregion for MDHV-A antigen within the MDHV genome. Initially the MDHV-Agene was identified within the library of pBR328 clones (FIG. 1)previously developed and studied in these laboratories (Gibbs, C., K.Nazerian, L. F. Velicer, and H. J. Kung, Proc. Natl. Acad. Sci. USA81:3365-3369 (1984)). The gene was first localized on a 2.35 kbpsubfragment of an approximately 6.8 kbp clone temporarily designatedpBR328-15 (FIG. 2) herein. The DNA fragment carrying the MDHV-A gene isactually the one designated M in other published work using these clones(Gibbs, C., K. Nazerian, L. F. Velicer, and H. J. Kung, Proc. Natl.Acad. Sci. USA 81:3365-3369 (1984)).

Further work with the MDHV-A gene in this clone seemed inadvisable fortwo reasons: 1) location of the 2.35 kbp subfragment near one end of thelarger fragment (FIG. 2) means it was possible that the entire gene wasnot present on the fragment, and 2) the pBR328 library contains only 30%of the MDHV genome. Thus, since none of the other pBR328 cloneshybrid-selected MDHV-A the real possibility existed that not all of itsgene was available. Fortunately at this time Fukuchi et al (Fukuchi, K.,M. Sudo, Y.-S. Lee, A. Tanaka, and M. Nonoyama, J. Virol. 51:102-109(1984)) published a paper on the structure of the MDHV genome using theGA strain, the same one used in our original cloning studies (Gibbs, C.,K. Nazerian, L. F. Velicer, and H. J. Kung, Proc. Natl. Acad. Sci. USA81:3365-3369 (1984)). These authors (Fukuchi, K., M. Sudo, Y.-S. Lee, A.Tanaka, and M. Nonoyama, J. Virol. 51:102-109 (1984)) presented Bam Hl,Bgl I and Sma l restriction endonuclease maps of MDHV DNA. They hadsuccessfully cloned 27 of the 29 Bam Hl fragments and these were thenmade available for study by others. Since their Bam Hl libraryconstituted nearly 100 percent of the MDHV genome and was well mapped itseemed wisest to use it as a potential source of the entire MDHV-A genefor all future studies.

Initial localization of the MDHV-A gene in the Bam HI library was bySouthern blot analysis with the 2.35 kbp Eco Rl-Pvu II subfragment ofthe pBR328 clone (FIG. 3) used as a probe of the entire set of Bam Hlfragments. Clearly the MDHV-A genetic material in the 2.35 kbpsubfragment hybridized only with the 18.3 kbp Bam Hl fragment B (FIG.3). Upon further restriction mapping and hybrid-selection analysis itwas found that the MDHV-A coding region lies on a 4.6 Pvu II-Sma lfragment found within the larger Bam Hl B fragment (FIG. 4). Becauseearly mapping studies revealed an Eco Rl site this fragment can besubdivided into a 2.35 kbp Eco Rl-Pvu II fragment, and a 2.4 kbp EcoRl-Sma l fragment, both of which can hybrid-select MDHV-A mRNA. However,the extent of selection is much greater with the former than with thelatter fragment (FIG. 4A). Nevertheless these data appear to confirm thevalidity of our original concern that the 2.35 kbp Eco Rl-Pvu IIsubfragment of the pBR328-15 clone might not have contained the entiregene. However the weak hybrid-selection with the 2.4 kbp Eco Rl-Sma Ifragment suggests that it contains only a very small piece of the MDHV-Agene, if any at all, a conclusion consistent with the very weak positiveresults obtained with the same fragment upon Northern blot analysis withlong exposure of the X-ray film (FIG. 4). However it is now believedthat these weak results are only due to a read-through duringtranscription of the gene. Nuclease Sl protection experiments wereconducted in an attempt to determine more precisely the extent of theMDHV-A gene on the 2.4 kbp Eco RI-Sma I fragment and it was not foundthere. Furthermore DNA sequencing studies will include this fragment aswell as to determine if it contains part of the MDHV-A gene's openreading frame. However, because of the inability of the othersurrounding subfragments to hybrid-select MDHV-A mRNA, or detect themRNA upon Northern blot analysis, it is assumed that the complete Aantigen coding region has been identified within the center of the BamHl B fragment. This fragment is in the right handed half of the uniquelong region of the virus genome based on the map of Fukuchi et al.(Fukuchi, K., M. Sudo, Y.-S. Lee, A. Tanaka, and M. Nonoyama., J. Virol.51:102-109 (1984)).

Based on the size of MDHV-A's unglycosylated precursor polypeptide, pr47(Isfort, R. J., R. A. Vrable, K. Nazerian, H.-J. Kung, and L. F.Velicer. Gene identification and molecular characterization of theMarek's disease virus A antigen, p. 130-147. In B. W. Calnek and J. L.Spencer (eds.), Proc. Int. Symp. Marek's Dis. The American Associationof Avian Pathologists, Inc, Kennett Square, Pa. (1984); Isfort, R. J.,R. A. Stringer, H. J. Kung, and L. F. Velicer, J. Virol. 57:464-474(1986), FIG. 1 and FIG. 2), it is possible to predict that its mRNAshould be approximately 1.7 kbp. Detection of MDHV-A mRNA of 1.8 kbp isconsistent with this prediction, and the small size difference suggeststhat splicing is minimal if it occurs at all.

So far, a function for the A antigen has not been found. Its possiblerole(s) in immunoprevention and/or immunosuppression were mentioned inthe introduction. Possibly it is not needed for virus growth since Aantigen negative strains are known and are able to propagate well intissue culture. However more rigorous proof that these strains are trulynegative may be called for. Recently however, the A antigen has beenimplicated as having a role in host immunoevasion, possibly as afunction of its extensive secretion (Isfort, R. J., R. A. Stringer H. J.Kung, and L. F. Velicer, J. Virol. 57:464-474 (1986)).

DETAILED ANALYSIS OF THE MDHV-A GENE

The MDHV-A gene containing 2.35 kbp EcoRI-Pvu II segment of the MDHV BamHI B fragment was subcloned into the plasmid pUC19 to generate plasmidp19MDA 2.35. This plasmid was deposited Mar. 11, 1987 under the terms ofthe Budapest Treaty with the American Type Culture Collection 10801University Boulevard, Manassas, Va. 20110-2209. The culture will beavailable to all who request it upon issuance of a patent as ATCC 40312.This subclone has been used to construct detailed maps of restrictionenzyme cleavage sites within the 2.35 kpb MDHV fragment. The 2.35 kbpfragment is cut at least once by the following restriction enzymes: BglII, EcoRI, EcoRV, Kpn I, Dra I, Dde I, Nco I, Ssp I, Spe I, and Taq I.

To unambiguously determine the orientation of the MDHV-A gene acombination of Northern blotting and nuclease protection analyses wasemployed. The 2.35 kbp EcoRI-Bam HI fragment of p19MDA2.35 was subclonedinto the multiple cloning site (MCS) of plasmid pGem4 (Promega Biotech,Madison, Wis.). This placed the 2.35 kbp fragment between opposing SP6and T7 RNA polymerase promoter sequences. Linearized plasmid DNA wasused with either SP6 and T7 polymerase to direct the transcription ofradioactively labeled complementary RNAs from the 2.35 kbp fragment.These labeled RNAs were used to probe Northern blots of MDHV-infectedcell mRNA. In this system, only that RNA probe which is identical to theactual MDHV coding strand will hybridize to MDHV-A mRNA. This samesystem can be used as detailed below to generate smaller ³² P labeledRNA probes (length determined by choice of linearizing enzyme) for usein a nuclease protection analysis using RNase A and RNase T1 tospecifically degrade unprotected RNA probe. In Northern blots only the³² P labeled SP6 generated probe hybridized to MDHV-A mRNA immobilizedon nitrocellulose, indicating that the MDHV-A gene was transcribed inthe direction shown in FIG. 4B. These results were supported by Slnuclease analysis using DNA probes (corresponding to I and II of FIG.4B). Plasmid p19MDA2.35 was digested with Bgl II and radioactivelylabeled with polynucleotide kinase and (-³² p) ATP. Further cleavagewith EcoRI and Bam HI generated probes I and II of FIG. 4B. Since theseprobes were labeled on different strands, only that strand which wascomplementary to the MDHV-A mRNA would hybridize to MDHV-A mRNA and beprotected from Sl nuclease digestion. Consistent with the results fromNorthern blotting with SP6 and T7 polymerase generated RNA probes, onlyprobe I was protected. Furthermore two species of probe I were protectedin the above experiment, a major species of approximately 530nucleotides and a minor species of 450 nucleotides thus indicating twopotential transcription initiation sites within the Bam HI-Bgl IIfragment, as indicated by solid arrows in FIG. 4B. The data from theseexperiments is being used to prepare smaller probes for additional Slnuclease protection and primer extension experiments. The combination ofSl, primer extension, and DNA sequence analyses will allow preciselocalization of the MDHV-A gene transcription start site(s).

The MDHV-A gene's 3' end has been mapped approximately 367 nucleotides5' of the EcoRI site (FIG. 4B, dashed arrow) by RNase A/T1 protectionanalysis using SP6 generated RNA probes (probes II and III, FIG. 4B).Once again location was further refined by traditional Sl nucleaseprotection analysis using a probe which extends from NcoI to EcoRV. Thegene's orientation and limits suggest that the small amount of MDHV-AmRNA hybrid-selected by the 2.4 kbp EcoRI-Sma I segment of the Bam HI Bfragment (FIG. 1) may be due to read-through during transcription of thegene.

This invention is extremely important to the MDHV system in threeways: 1) this is the first gene identified and cloned in this system,and 2) the means now exist to use modern approaches of recombinant DNAtechnology to study and use this late gene and its glycoprotein product.It is now known MDHV-A is a late protein based on phosphonacetic acidstudies (L. F. Velicer, unpublished data). Thus these current studiesshould shed much light on the nature of a gene for a unique lateglycoprotein antigen that is rapidly secreted from DEF cells, but mayalso have a membrane component (Isfort, R. J., R. A. Stringer H. J.Kung,and L. F. Velicer. J. Virol. 57:464-474 (1986). As the inventionprogresses it is expected more will be learned about the nature of anoncogenic herpesvirus encoded antigen that may be involved inimmunoprevention, immunosuppression and/or possible immunoevasion.

The MDHV-A gene has a significant usefulness for diagnostic purposes intwo different ways. First, the gene itself can be used directly forhybridization-based diagnostics to detect Marek's disease herpesvirus inbirds, since it is also present in pathogenic Marek's diseaseherpesviruses. For example, as broilers are shipped to market at anearlier age infected birds might arrive for slaughter before lesionsappear (the basis for condemnation) and the poultry industry may need arapid diagnostic technique to screen flocks for infection.

Also when recombinant DNA vaccines are on the market it will benecessary to differentiate those of an infectious nature (one of manyvaccine forms under consideration) from the wild type virus that isspread in nature, especially for legal and environmental reasons. Inthis case the MDHV-A gene is useful to modify slightly (e.g., insertsome sort of distinctive marker DNA sequence) so the recombinant vaccinevirus can be readily identified.

The MDHV-A gene is useful in the development of sensitive and specificELISA based diagnostics of an immunologic nature, in one of two ways,based on production of MDHV-A in cells transfected with this gene andproducing the antigen. The antigen can be used directly in an ELISAassay to detect antibody in the sera of chickens. It could also be usedindirectly to produce highly specific antibody (monoclonal andpolyclonal) that can be used on the ELISA assay to detect the antigen ininfected birds. The A antigen is of special interest in terms ofdiagnostic sensitivity because it is the most prominent antigen madeduring infection, and it is also the one the bird reacts to mostextensively. Thus it would be our first choice for diagnostic purposes.

On top of all else MDHV-A has a signal peptide which facilitates theantigen's secretion from cells. The part of the gene encoding its signalpeptide may be extremely valuable in the production of the otherantigen(s). If this part were put onto the gene encoding another antigenthat is not normally secreted, the other antigen might now be secreted.This would be an extremely valuable feature in the production andpurification of this other antigen in eucaryotic expression vectors.There is also a promoter which can be used to promote the expression ofunrelated genes. Thus the signal peptides and promotors are used asregulatory elements for other genes.

It is intended that the foregoing description be limited only by thehereinafter appended claims.

What is claimed is:
 1. A purified EcoRl-PvulI restriction subfragmenthaving a length of about 2.35 kbp in a BAM HI B restriction fragment ofMarek's disease herpesvirus genome wherein the subfragment encodes Aantigen precursor.
 2. A plasmid comprising the subfragment of claim 1.3. An expression vector comprising the subfragment of claim
 1. 4. A cellwith an expression vector comprising the subfragment of claim 1 whichcell expresses A antigen precursor because of the presence of thesubfragment.
 5. The plasmid of claim 2 wherein the plasmid is pUC19. 6.A gene fragment encoding A antigen precursor of Marek's diseaseherpesvirus which is an insert in plasmid pUC19 deposited as ATCC 40312.7. A purified gene fragment coding for Marek's disease herpesvirus Aantigen precursor which is a EcoRl-PvuII endonuclease insert in pUC19deposited as ATCC
 40312. 8. A method for isolating the gene coding for Aantigen precursor from DNA of Marek's disease herpesvirus whichcomprises:(a) digesting a BAM HI B fragment of the DNA with EcoRl-PvuIIendonucleases to provide a subfragment having a length of about 2.35 kbpwherein the subfragment encodes the A antigen precursor; and (b)isolating the subfragment, which codes for the A antigen precursor byhybrid selection with MDHV-A mRNA.